Project Abstract Many Americans have tried drugs of abuse at some point during their lives, but only 3% of the population is classified as having a substance abuse disorder. Not all drug use develops into a disorder; however, the mechanisms underlying this switch remain unknown. This proposal will examine the changes in specific neuronal ensembles and perineuronal nets (PNNs) in the medial prefrontal cortex (mPFC) between an initial drug-taking experience and later drug seeking. Neuronal ensembles are sparsely distributed sets of neurons that are responsible for manifestation of a specific behavior. PNNs are structures in the extracellular matrix surrounding a subset of neurons within the brain that develop and adapt in an activity-dependent manner. Our central hypothesis is that a persistent drug-seeking ensemble is formed in the mPFC at the time of the initial experience which is solidified by the PNNs surrounding ensemble neurons. We also hypothesize that the proportion of ensemble neurons reactivated during a later drug-seeking experience will predict the degree of drug-seeking behavior. We will test these hypotheses by 1) identifying the overlap and output within mPFC ensembles during an initial opioid-taking experience and future opioid seeking, and 2) defining the characteristics of PNNs surrounding mPFC ensembles during an initial opioid-taking experience and future opioid seeking. We will use a novel method of single-phase, self-administration learning to serve as the initial experience which allows us flexibility to study variability in initial use and the later consequences. Additionally, we will use double-transgenic TetTag mice to allow for the identification of neuronal ensembles at two distinct time points. The studies proposed here will have a lasting impact on both the neuronal ensemble and PNN fields by providing more clarity into the plasticity contributing to learning and memory of an opioid experience which may facilitate the progression from drug use to drug abuse.